Blocking the alternative NF-κB pathway with anti-BR3 antibody. (A) CLL B cells were cultured for 24 hours with or without rhBAFF (50 ng/mL) and anti-BR3 at the indicated concentrations. Cytoplasmic and nuclear extracts were prepared as described in “Materials and methods” for immunoblot analysis. Protein content was normalized to 25 μg for the cytoplasmic fraction and 12.5 μg for the nuclear fraction. MFIR of CLL cells stained for BCMA, TACI, or BR3 was 3.7, 8.8, or 24.3, respectively. Translocation of p52 and p65 to the nucleus was seen in CLL cells treated with rhBAFF. Anti-BR3 at 10 μg/mL could completely inhibit nuclear translocation of p52 induced by rhBAFF. (B) CLL B cells were cultured with or without rhBAFF (50 ng/mL), anti-BR3 (10 μg/mL), BR3-Fc (10 μg/mL), or control immunoglobulin (10 μg/mL) for 24 hours. Nuclear extracts and total cell lysates were prepared as described in “Materials and methods.” BR3-Fc inhibited the rhBAFF-induced nuclear translocation of p52 and the phosphorylation of IκBα. Anti-BR3 could inhibit the nuclear translocation of p52 but not the phosphorylation of IκBα. (C) CLL B cells were cultured with or without rhBAFF (50 ng/mL) and anti-BR3 (10 μg/mL) or BR3-Fc (10 μg/mL) for 48 hours. Results are viability of samples from each of 5 patients. The viability of CLL cells cultured with rhBAFF and BR3-Fc was significantly lower than that of CLL cells cultured with rhBAFF alone (P < .001; Student paired t test). Anti-BR3 did not impair the survival of CLL cells cultured with rhBAFF.